At present, it is known that various factors may effect the efficiency of expression of eucaryotic proteins whose genes have been cloned onto bacterial plasmids or expression vectors. Two of the most important factors which both define and govern this protein expression are the accuracy with which the bacterial cell transcribes the inserted gene into mRNA and the efficiency of translation of this mRNA by bacterial ribosome into protein. The efficiency of transcription and translation, generally known as expression, is believed to be dependent upon the nucleotide sequences typically situated ahead of the cloned genes on the vector DNA. These expression control sequences, in large part, comprise promoter/operator sites wherein the RNA polymerase interacts to initiate transcription and ribosome binding sites wherein the ribosomes bind and interact with the mRNA to initiate translation into protein.
The prior art has uncovered varous obstacles to expressing eucaryotic proteins whose genes have been cloned onto bacterial plasmids. For instance, the prior art has recognized that it may be both harmful to the host cell and the stability of the hostvector system if the eucaryotic protein is expressed and is present in the cell in large quantities. The prior art has also recognized that the mRNA's transcribed in a bacterial host from cloned eucaryotic DNA inserts do not possess the necessary regulatory signals, i.e., ribosome binding sites, (as do procaryotic mRNA's) for recognition by procaryotic ribosomes.
In order to overcome these potential obstacles, the prior art has attempted to alter or substitute the naturally occurring portion of the nucleotide sequences which control or regulate the transcription and/or translation process or ribosome binding process. A tailored expression control sequence, desired by the prior art, in addition to improving the efficiency of transcription and translation of the cloned genes, should also be controllable so as to modulate expression during bacterial growth. The expression control sequences preferred by the prior art to date are ones that may be selectively switched on and off. Host cells are thereby allowed to propagate without excessive build-up of gene products prior to switching on so as to promote the expression of large amounts of the desired protein products.
Various expression control sequences, different from those naturally occurring on the plasmid have, therefore, been employed by the prior art to improve control over the expression of eucaryotic proteins and polypeptides in bacterial hosts. These include, for example, utilization of the operator, promoter, ribosome binding and interaction sequences of the lactose operon of E. coli, the corresponding sequences of the tryptophan synthetase system of E. coli and the major operator and promoter regions of bacteria phage .lambda. (a bacterial DNA virus) [H. Bernard et al., "Construction Of Plasmid Cloning Vehicles that Promote Gene Expression From The Bacteriophage Lambda P.sub.L Promoter", Gene, 5, pp. 59-76 (1979) said reference being incorporated by reference herein.
The P.sub.L promoter of the .lambda. phage has been used to express eucaryotic and procaryotic proteins cloned into a procaryotic organism. The promoting activity of P.sub.L was found to be switched off at low temperature in the presence of a cI(ts) temperature sensitive gene that specifies a temperature-sensitive repressor, but could be activated by heat induction when expressing a procaryotic protein. Examples of the prior art use of this promoter system may be seen in European Patent Application 81301413.1 filed Apr. 1, 1981 and Derynck et al., "Expression of human fibroblast interferon gene in Escherichia coli", Nature, 287, 193-197 (1980). In these references, both of which are incorporated by reference herein, the P.sub.L promoter system was used in conjunction with the entire bacterial ribosome binding site in expressing eucaryotic proteins such as fibroblast interferon.
In order to express eucaryotic genes in E. coli the prior art has generally taken one of two approaches, both utilizing recombinant DNA techniques, so as to overcome the lack of proper regulatory signals on the eucaryotic genes: (1) inserting the eucaryotic coding sequence within the coding region of a bacterial gene, such as .sym.-lactamase; thus utilizing the entire ribosome binding site (RBS) of the E. coli gene, or (2) constructing a so-called "hybrid" RBS consisting of the SD sequence, the sequences proximal to the SD sequence (from the E. coli DNA and AUG start codon) plus the remaining coding sequence derived from the eucaryotic DNA. The SD sequence is a sequence of 3 to 9 bses which are complementary to bases at the 3'-end of 16s ribosomal RNA known as the Shine-Delgarno sequence (hence, SD) which bases are located between the promoter and initiation codon for the eucaryotic gene. One or the other of these approaches is necessary for expression of eucaryotic DNA in E. coli since eucaryotic genes do not contain sequences resembling the SD sequence at the start of translation.
The major disadvantage of the first approach is that it results in the production of a fusion polypeptide, that is, part of the gene product is encoded by the E. coli DNA and part by the eucaryotic DNA. This fusion protein may however, be processed into active form by unknown and inefficient mechanisms within the cell. The second approach has been successfully employed to produce authentic mature (complete and biologically active with no attached pre-sequence) gene products identical to those produced in the natural situation, i.e., in the eucaryotic cell. See Derom et al., "High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda P.sub.L promoter", Gene, 17, 55-54 (1982); and Gheysen, et al., "Systematic alteration of the nucleotide sequence preceding the translation initiation codon and the effects on bacterial expression of the cloned SV40 small-t antigen gene" Gene, 17, 55-63 (1982) both references being incorporated by reference herein.
To date, the prior art has attempted to express various human interferon species, such as leukocyte, fibroblast and immune, with both approaches. While attempting to express fibroblast interferon (FIF), the aforementioned Gheysen et al reference utilized the first approach (wholly bacterial RBS) in conjunction with the P.sub.L promoter system. Although this reference indicates that biologically active, non-fusion proteins were obtained, it is clear that this was a result of cellular processing of fusion FIF proteins by unknown and inefficient mechanisms within the cell since the FIF gene does not have the proper translation initiation sequences. It is further apparent from this reference that the eucaryotic gene product contains the amino-acids sequence coded by the signal portion of the eucaryotic gene. Therefore, a mature form of interferon is not being expressed.
In the following references, (both being incorporated by reference herein) Gray, et al., "Expression of human immune interferon cDNA in E. coli amd monkey cells", Nature, 295, 503-508 (1982), ahd Shepard et al., "Increased Synthesis in E. coli of Fibroblast and Leukocyte Interferons Through Alterations in Ribosome Binding Sites" DNA, 1, 125-131 (1982), the "hybrid RBS" second approach was utilized. However, as noted in these references, expression was under the control of the E. coli trp promoter and the hybrid RBS were derived from the ATG of the eucaryotic gene and the SD and linker sequence of the trp leader. Furthermore, in both of these references the SD sequence and sequence proximal (upstream) to the SD sequence remained unchanged. The only variability introduced was into the linker region, that is a change in both number and composition of the nucleotides between the SD sequence and the ATG codon.
The prior art lacked a means for designing and constructing a hybrid ribosome binding site with the flexibility required to selectively increase expression of a cloned eucaryotic gene. Furthermore, the prior art lacked a means for controlling the expression of a cloned eucaryotic gene protein in its mature form, with a temperature sensitive promoter system and without the need for independent cellular processing of fusion proteins.